This invention relates to a method for long-term refrigerated storage of red cells.
Red cells stored by refrigeration have a limited shelf-life depending on the solutions in which they are stored. Shelf-life is determined, in the United States at least, by measurements of the proportion of cells circulating in the recipient 24-hrs after transfusion. The FDA has unofficially established 75% as the minimum for a licensed product. The quantity of free hemoglobin that is transfused can also limit shelf-life. Although no official maximum has been established, there is general agreement that hemolysis should not exceed 1%.
The two general methods for the refrigerated storage of human red blood cells are: (1) refrigerated storage in the original anticoagulant solution; and (2) refrigerated storage after separation of the red cells from the original anticoagulant solution, and resuspension of the cells in a solution that is specifically designed for red cell storage. When either of these methods is used, at least a residual amount of plasma remains in the red cell solution.
For storage in the original anticoagulant, whole blood is conventionally drawn into a solution containing citrate, phosphate, dextrose (d-glucose) and adenine (CPDA-1). The blood is centrifuged at about 1500.times.G (soft spin) and the plasma is removed, leaving a red cell suspension with a hematocrit of about 75%. Platelets can be removed from the plasma by a second sedimentation. Cells collected in CPDA-1 can be stored for 5 weeks without further treatment with or without removal of the plasma.
For resuspension of the red cells and storage in a preservation solution, blood is conventionally drawn into a solution containing only citrate, phosphate and glucose. The blood is centrifuged at about the same speed as described above but the red cells are then resuspended in approximately 100 ml of an additive solution, resulting in a red cell suspension at a hematocrit of approximately 5%. The two currently-licensed additive solutions in the United States are Adsol and Nutricel, as defined in Table 1. Another known additive solution, Sagman, has not been licensed in the United States. Cells stored with these additive solutions have a six-week shelf-life.
TABLE 1 ______________________________________ CPDA-1 ADSOL NUTRICEL Ingredient (mM) (mM) (mM) ______________________________________ NaCitrate 89.6 -- 20.0 cit. acid 15.6 -- 2.0 dextrose 161.0 111.0 55.5 NaH.sub.2 PO.sub.4 16.1 -- 20.0 Adenine 2.0 2.0 2.2 Mannitol -- 41.2 -- NaCl -- 154.0 70.1 Osmolality 323 342 244 (mOsm) pH 5.7 5.5 5.8 ______________________________________ CPDA-1 and ADSOL are sold by Baxter Travenol and NUTRICEL is sold by Cutter. Osmolality is the effective osmolality contributed by the nonpenetrating constituents.
During storage, human red blood cells undergo morphological and biochemical changes, including decreases in the cellular level of adenosine triphosphate (ATP) and 2,3-diphosphoglycerate (2,3-DPG), changes in cellular morphology, and progressive hemolysis. The concentration of ATP, after a brief initial rise, progressively declines to between 30 and 40% of its initial level after six weeks of storage. The fluidity of the cell membrane of red cells, which is essential for the passage of red cells through the narrow channels in the spleen and liver, is loosely correlated with the level of ATP.
The primary function of red cells in the circulation is to deliver oxygen to the tissues. A unique characteristic of hemoglobin is that it can unload much of its oxygen even though the partial pressure of oxygen in the tissues may be relatively high. A compound called 2,3-diphosphoglycerate (2,3-DPG) is essential to this process and, in its absence, oxygen is not efficiently delivered to the tissues. During refrigerated storage as currently practiced, the level of 2,3-DPG falls rapidly after about three or four days of storage and approaches zero by about ten days.
Morphological changes occur during storage, ultimately leading to the development of spicules on the red cells (echinocytosis). These spicules can bud off as vesicles, radically changing the surface-to-volume ratio of the cells and their ability to deform on passing through narrow channels. Such cells will be filtered out of the circulation by the spleen and liver following transfusion. As stated above, to be acceptable for transfusion at least 75% of the red cells that are transfused must be capable of remaining in circulation twenty-four hours following the transfusion. The concentration of ATP and the morphology of red cells serve as indicators of the suitability of stored cells for transfusion.
In order to prolong the shelf-life of transfusable red blood cells, it is necessary to store the cells or treat them in some manner that prevents a rapid decline in ATP and, if possible, 2,3-DPG. Solutions that prolong the shelf-life of red cells are known (see, e.g., Meryman, U.S. Pat. No. 4,585,735, and Meryman, U.S. Pat. No. 5,250,303, both of which are herein incorporated in their entirety by reference). Typically such solutions contain citrate, phosphate, glucose and adenine and occasionally other ingredients that function to prolong shelf-life by maintaining the level of ATP in the cells. It is known to use an additive solution having an effective osmolality as low as 121 mOsm. However, solutions with lower effective osmolalities are not used. In addition, glycolytic activity is enhanced in red blood cells if the intracellular pH (hereinafter ph.sub.i) measured at 4.degree. C. is about 7.4.
The effective osmolality of the suspending solution is another factor of importance in extending red cell storage time. It has been shown that effective hypotonicity substantially reduces hemolysis and improves red cell morphology during storage. Although the mechanism has not been proven, it is probable that osmotic swelling increases cell surface tension, thereby facilitating the shape changes usually associated with stored red cells.
When red cells are washed, it is possible to achieve the maximum hypotonicity just short of hemolysis from cell swelling. However, washing red cell units is expensive and not currently justified by the extended shelf-life obtained. The standard procedure currently in use involves the removal of plasma following an initial sedimentation and the addition of 100 ml of an additive solution to approximately 200 ml of red cells and 50 ml of residual plasma.
The hypotonicity of the additive solution is limited by the danger of hemolysis during the addition of the solution. Although, theoretically, it is not necessary for the additive solution to contain enough solute to osmotically support the red cells since the plasma provides additional osmotic support, at the time of adding the solution, before mixing has occurred, some red cells will come into contact with the additive solution. If the additive solution is too hypotonic, these red cells will burst (hemolyze). As a result, solutions that are too hypotonic cannot be used. Therefore, the final osmolality of the solution after mixing with the cells and the residual plasma is not particularly hypotonic and the advantages of hypotonicity are insufficient.
Red cells, which are normally bi-concave disks, can swell to nearly twice their normal volume at an external osmolality of approximately 170 mOsm before they hemolyze. However, it has also shown that when solutes from the left end of the Hofmeister series, the so-called macromolecular stabilizers, are present both inside and outside the cell, a membrane expansion takes place and red cells can swell beyond their normal hemolytic volume and do not begin to hemolyze until the extracellular osmolality is approximately 70 mOsm. Meryman, H.T., "Influence of certain neutral solutes on red cell membrane area and permeability during hypotonic stress," Am. Journ. of Physiol., 225:365-371, 1973.
Because of the critical need for transfusable red blood cells, it is of great importance not only to develop methods and solutions that not only maintain high intracellular levels of both ATP and 2,3-DPG, good morphology and low hemolysis after washing, but also to develop methods for the routine collection and resuspension of unwashed red cells with better storage characteristics than are achieved by current procedures.